Early detection of disease states in mammals has been the focus of much recent research. For disease detection, the public-health community has historically relied on laboratory tests that can sometimes take days or even weeks to return a result. The increased availability of better and faster diagnostic tests, however, promises the possibility of more automated and earlier disease detection and subsequent intervention. It is believed that introduction of therapy early in the disease process will reduce the mortality rate associated with the disease and shorten the time for treatment.
Acute renal failure (ARF) secondary to renal injury, including but not limited to ischemic injury and nephrotoxic injury, remains a common and potentially devastating problem in clinical nephrology. Five percent (5%) of hospital admissions and 30% of Intensive Care Unit admissions have acute renal failure, and 2-5% of hospitalized patients will develop it. Acute renal dysfunction occurs in up to 40% of adults following cardiac surgery. Pathophysiologic mechanisms include diminished renal blood flow, loss of pulsatile flow, hypothermia, atheroembolism, and a generalized inflammatory response. ARF requiring dialysis also complicates up to 10% of cardiac surgeries in infants and children with congenital heart disease.
ARF persistently continues to result in a high rate of mortality despite significant advances in supportive care. Pioneering studies over several decades have illuminated the roles of persistent vasoconstriction, tubular obstruction, cellular structural and metabolic alterations, and the inflammatory response in the pathogenesis of ARF. While these studies have paved the way for successful therapeutic approaches in animal models, translational research efforts in humans have yielded disappointing results, for reasons such as the multifaceted response of the kidney to ischemic injury and a paucity of early markers for ARF with a resultant delay in initiating therapy.
Animal studies have shown that, while ARF due to ischemia can be prevented and/or treated by several maneuvers, treatment for ARF must be instituted very early after the ischemic insult. A major reason for the inability to provide preventive and therapeutic measures for ARF in humans is the lack of early biomarkers for ARF. Thus, the identification of a reliable, early biomarker for impaired renal status would be useful to facilitate early therapeutic intervention, and help guide pharmaceutical development by providing an early indicator of nephrotoxicity.
The traditional laboratory approach for detection of renal disease involves determining the serum creatinine, blood urea nitrogen, creatinine clearance, urinary electrolytes, microscopic examination of the urine sediment, and radiological studies. These indicators are not only insensitive and nonspecific, but also do not allow for early detection of the disease. In current clinical practice, ARF is typically diagnosed by measuring a rise in serum creatinine over time, which is an unreliable indicator for measuring acute changes in kidney function. Indeed, while a rise in serum creatinine is widely considered as the “gold standard” for the detection of ARF, it is a late indicator of renal injury since as much as 50% of the kidney function may already be lost by the time the serum creatinine changes. Currently there are no tools available for the early diagnosis of impaired renal status.
The lack of early biomarkers for acute renal injury thus has severely slowed progress in finding effective therapies within the narrow window of opportunity. The identification of urinary protein biomarkers suitable for the early detection and diagnosis of acute renal injury holds great promise to improve the clinical outcome of patients. It is especially important for patients presenting with vague or no symptoms or with acute renal injury following surgery such as cardio-pulmonary bypass surgery. Despite considerable effort directed at early detection of ARF, no cost-effective screening tests have been developed to date.
Although efforts to evaluate disease processes and drug effects have traditionally focused on genomics, more attention has been paid recently to proteomics due to its offering a more direct, complete and promising understanding of the biological functions of a cell. The term “proteomics” was coined to make an analogy with genomics, and while it is often viewed as a continuation of genomics, proteomics is much more complicated than genomics. Most importantly, whilst the genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. One organism will have radically different protein expression in different parts of its body, in different stages of its life cycle and in different environmental conditions.
The protein map of a biological system, including a cell, sub-cellular fraction or expression media, can be referred to as a proteome. Proteomics, or analysis of the proteome of a biological system, offers a relatively new approach to protein expression profiling and cellular or tissue protein identification from samples that are obtained under various specified conditions. Proteomics has an enormous breadth of application ranging from investigation and identification of biomarkers, molecules that are indicative of a particular pathological state, which in turn can be used for diagnostic purposes and targets for therapeutic intervention. Proteome analysis allows the investigator to obtain information on protein identity, protein-protein interaction, the level of protein expression and protein expression profiling, protein trafficking and turnover, protein variants, and protein post-translational modifications.
Traditionally, proteomics combines two-dimensional electrophoresis (2-DE), a high-resolution protein separation technique, with mass spectrometry (MS). Proteomics research is targeted towards characterization of the proteins encoded by a particular genome and its changes under the influence of biological stimulation. Proteomics also involves the study of non-genome encoded events such as the post-translation modification of proteins, interactions between proteins, and the location of proteins within the cell. The study of gene expression at the protein level is important because many of the most important cellular activities are directly regulated by proteins in the cell rather than by gene activity. Also, the protein content of a cell is highly relevant to drug discovery and drug development efforts since most drugs are designed to target proteins. Therefore, the information gained from proteomics is expected to greatly boost the number of drug targets.
Attempts at unraveling the molecular basis of early renal responses have been facilitated by recent advances in functional genomics that have yielded new tools for genome-wide analysis of complex biologic processes. To date, the most popular method for proteomics investigation is the use of high-resolution two-dimensional gel electrophoresis and sensitive mass spectrophotometry techniques. Although two-dimensional gel electrophoresis is one of the most powerful methods in the current study of proteomics, this method is labor-intensive, time consuming, and limited in sensitivity. The two-dimensional gel electrophoresis method also suffers from poor reproducibility. To avoid the aforementioned disadvantages of two-dimensional gel electrophoresis, microchip-based separation devices (microarrays) have been developed for rapid analysis of large numbers of samples. Compared to conventional separation columns or devices, microarrays have higher sample throughput, reduced sample and reagent consumption, and reduced chemical waste. Such devices are capable of fast analyses and provide improved precision and reliability compared to the conventional analytical instruments. The cDNA microarray methodologies provide parallel and quantitative expression profiles of thousands of genes, which when combined with bioinformatics tools, can identify genes in a biologic pathway, characterize the function of novel genes, and detect disease subclasses. However, until now, no early stage molecular markers have been identified for ARF.
Mass spectrometry is a technique that measures m/z (mass-to-charge) intensity pairs of an ionizable substance. The m/z-intensity pair or pairs of an analyte provides a signature distinguishing the analyte from other substances having a different m/z-intensity pair or pairs. The intensity of an analyte's m/z-intensity pair changes with the analyte's abundance within the response range of the instrument. Techniques and equipment for generating mass spectrometry data are well known in the art. Examples of ionization techniques that can be employed include electronspray ionization, matrix-assisted laser desorption/ionization (MALDI), surface enhanced laser desorption/ionization (SELDI), electron impact ionization, chemical ionization, and photoionization.
Recently, a chip-based proteomics approach has been introduced using biomolecular interaction analysis-mass spectrometry (BIA-MS) in rapidly detecting and characterizing proteins present in complex biological samples at very low levels. One of the most powerful techniques is Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) technology, which has been commercially embodied in Ciphergen's ProteinChip® Biomarker System. The system uses chemically (cationic, anionic, hydrophobic, metal, etc.) or biochemically (antibody, DNA, enzyme, receptor, etc.) treated surfaces for specific interaction with proteins of interest, followed by selected washes for SELDI-TOF-MS detection. Surface-Enhanced Laser Desorption/Ionization (SELDI) was invented in the late 1980's. When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides a means to rapidly analyze molecules retained on a chip. The power of the system incorporates straightforward sample preparation with on-chip capture (binding) and detection for protein discovery, protein purification, and protein identification from small samples, allowing rapid analysis and assay development on a single platform.
Several tubular proteins have been measured in the urine, with conflicting and unsatisfactory results. For example, one cDNA microarray expression profile identifies kidney injury molecule-1 (KIM-1), a novel kidney-specific adhesion molecule involved in renal regeneration which is upregulated 24-48 hours after initial insult. KIM-1 is a reliable but somewhat late stage marker of tubular cell damage, and has been detected in the kidney biopsy and in the urine of patients with established ischemic acute tubular necrosis. However, this detection was documented in patients with established ischemic renal damage, late in the course of the illness. The utility of urinary KIM-1 measurement for the detection of early ARF or subclinical renal injury has thus far not been validated. Also, sodium-hydrogen exchanger isoform 3 (NHE-3) has been shown in the urine from subjects with established ARF. The sensitivity and specificity of these biomarkers for the detection or prediction of impaired renal status have not been reported. Of the inflammatory cytokines involved in ARF, elevated levels of urinary IL-6, IL-8 and IL-18 have been demonstrated in patients with delayed graft function following cadaveric kidney transplants. None of these biomarkers have been examined prospectively for their appearance in the urine during the evolution of ischemic ARF.
There is currently a lack of a reliable biomarker for the early determination of renal injury and disease caused by ischemia and/or nephrotoxicity. Therefore, it would be advantageous to provide testing of a subject's urine, blood serum, or other body fluid samples for early biomarkers of acute renal injury within minutes of a suspected injury, since early biomarkers for acute renal failure may begin to appear at low levels and continue to rise thereafter. It would likewise be advantageous if early biomarkers for acute renal injury could be detected in bodily fluid samples such as blood serum and urine shortly after the onset of a renal event that could lead to renal tubular cell injury. It would also be advantageous to use the ability of the SELDI-TOF-MS technology to rapidly identify protein biomarkers in a method of rapid identification of early biomarkers of various diseases, including ischemic and nephrotoxic renal injuries. There is also a need to provide a reliable and accurate method of early determination of the existence of acute renal injury in patients, the results of which can then be used to manage the treatment of affected patients.